title: Comparison of high-resolution melting analysis, TaqMan Allelic discrimination assay, and sanger sequencing for Clopidogrel efficacy genotyping in routine molecular diagnostics.
abstract: Clopidogrel, as a routine antiplatelet drug, is widely used in patients to reduce cardiovascular events following percutaneous coronary intervention. Because of genetic variation, patients undergoing percutaneous coronary intervention show differing responses to clopidogrel therapy. Recently, five single nucleotide polymorphisms (SNPs) within CYP2C19 (rs4244285, rs4986893, rs12248560), ABCB1 (rs1045642), and ITGB3 (rs5918) were identified that contribute prominently to variability in response to clopidogrel. Given that Sanger sequencing is labor intensive and time consuming, rapid genotyping methods for SNP detection are urgently required before clopidogrel therapy. Accordingly, we developed a high-resolution melting analysis (HRMA) and TaqMan allelic discrimination assay (TaqMan) to genotype those five SNPs, and compared these two assays with Sanger sequencing on accuracy of genotyping as well as operational characteristics. These two assays showed high accuracy (0.995, 95% CI 0.991 to 0.998 for HRMA; 0.997, 95% CI 0.994 to 0.999 for TaqMan, respectively), sensitivity (0.996, 95% CI 0.989 to 1.002 for HRMA; 0.998, 95% CI 0.993 to 1.002 for TaqMan, respectively), and specificity (0.995, 95% CI 0.991 to 0.999 for HRMA; 0.996, 95% CI 0.993 to 1.000 for TaqMan, respectively). Our study indicates that HRMA and TaqMan are easier to operate and obviously faster than Sanger sequencing. In conclusion, HRMA and TaqMan are rapid, convenient, and reliable assays for clopidogrel efficacy genotyping.
authors.affiliation: Institute of Hypertension and Department of Internal Medicine, Genetic Diagnosis Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China.
authors.forename: Lina
authors.initials: L
authors.lastname: Zhang
authors.forename: Guanglin
authors.initials: G
authors.lastname: Cui
authors.forename: Zongzhe
authors.initials: Z
authors.lastname: Li
authors.forename: Haoran
authors.initials: H
authors.lastname: Wang
authors.forename: Hu
authors.initials: H
authors.lastname: Ding
authors.forename: Dao Wen
authors.initials: DW
authors.lastname: Wang
mesh_terms: D000328:Adult; D000483:Alleles; D000077144:Clopidogrel; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods / Q000592:standards; D058885:Multilocus Sequence Typing / Q000379:methods / Q000592:standards; D010597:Pharmacogenetics / Q000379:methods; D010975:Platelet Aggregation Inhibitors / Q000627:therapeutic use; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013988:Ticlopidine / Q000031:analogs & derivatives / Q000627:therapeutic use; D016896:Treatment Outcome
chemical_list: D010975:Platelet Aggregation Inhibitors; D000077144:Clopidogrel; D013988:Ticlopidine